title: Characterization, gene cloning, and heterologous expression of β-mannanase from a thermophilic Bacillus subtilis.
abstract: Bacillus subtilis BCC41051 producing a thermostable β-mannanase was isolated from soybean meal-enriched soil and was unexpectedly found to be thermophilic in nature. The extracellular β-mannanase (ManA) produced was hydrophilic, as it was not precipitated even with ammonium sulfate at 80% saturation. The estimated molecular weight of ManA was 38.0 kDa by SDS-PAGE with a pi value of 5.3. Optimal pH and temperature for mannan-hydrolyzing activity was 7.0 and 60°C, respectively. The enzyme was stable over a pH range of 5.0-11.5, and at temperatures of up to 60°C for 30 min, with more than 80% of its activity retained. ManA was strongly inhibited by Hg(2+) (1 mM), but was sensitive to other divalent ions to a lesser degree. The gene of ManA encoded a protein of 362 amino acid residues, with the first 26 residues identified as a signal peptide. High expression of recombinant ManA was achieved in both Escherichia coli BL21 (DE3) (415.18 U/ml) and B. megaterium UNcat (359 U/ml).
authors.affiliation: Department of Biotechnology, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand.
authors.forename: Pijug
authors.initials: P
authors.lastname: Summpunn
authors.forename: Suttidarak
authors.initials: S
authors.lastname: Chaijan
authors.forename: Duangnate
authors.initials: D
authors.lastname: Isarangkul
authors.forename: Suthep
authors.initials: S
authors.lastname: Wiyakrutta
authors.forename: Vithaya
authors.initials: V
authors.lastname: Meevootisom
mesh_terms: D001410:Bacillus megaterium / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D002413:Cations, Divalent / Q000378:metabolism; D005591:Chemical Fractionation; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007526:Isoelectric Point; D008628:Mercury / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021382:Protein Sorting Signals; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D021382:Protein Sorting Signals; D044902:beta-Mannosidase; D008628:Mercury